Protocol for the Systematic Fixation, Circuit-Based Sampling, and Qualitative and Quantitative Neuropathological Analysis of Human Brain Tissue.

Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer's disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.

A Method to Visualize the Nanoscopic Morphology of Astrocytes In Vitro and In Situ.

In recent years it has become apparent that astroglia are not only essential players in brain development, homeostasis, and metabolic support but are also important for the formation and regulation of synaptic circuits. Fine astrocytic processes that can be found in the vicinity of synapses undergo considerable structural plasticity associated with age- and use-dependent changes in neural circuitries. However, due to the extraordinary complex, essentially nanoscopic morphology of astroglia, the underlying cellular mechanisms remain poorly understood.Here we detail a super-resolution microscopy approach, based on the single-molecule localisation microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM) to visualize astroglial morphology on the nanoscale. This approach enables visualization of key morphological changes that occur in nanoscopic astrocyte processes, whose characteristic size falls below the diffraction limit of conventional optical microscopy.

3D d STORM Imaging of Fixed Brain Tissue.

Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20-30 nm lateral and 50-70 nm axial resolution within an area of 500 µm × 500 µm and a depth of 0.6 µm to several micrometers. We employ single-molecule localization microscopy (SMLM) on immunolabeled fixed brain tissue slices. tomoSTORM utilizes array tomography to achieve SMLM in 40 nm thick resin-embedded sections. dSTORM of cryo-sectioned slices uses optical sectioning in 0.1-4 µm thick hydrated sections. Both approaches deliver 3D nanolocalization of two or more labeled proteins within a defined tissue volume. We review sample preparation, data acquisition, analysis, and interpretation.